The curative potential of allogeneic stem cell transplantation (alloSCT) is due largely to immunocompetent donor T cells that induce a potent graft-versus-tumor (GVT) response against host malignant cells. GVT is clinically effective against a number of hematologic malignancies, but is frequently accompanied by potentially fatal graft-versus-host disease (GVHD). Minor histocompatibility antigens (mHAgs) are MHC-bound peptides derived from normal cellular proteins that differ between alloSCT donor and recipient due to allelic amino acid polymorphisms. MHAgs form the basis of immunological non-identity between HLA- matched alloSCT donor and recipient, inducing high avidity T cell responses that mediate GVT, GVHD, or both. At present, only a rudimentary understanding of the mHAg targets of GVT and GVHD exists, due to the difficulty of identifying mHAgs with T cell clones derived from alloSCT patients. However, the molecular characterization of human mHAgs has suggested potential strategies to harness GVT and segregate it from GVHD by targeting for immunotherapy mHAgs that are selectively presented on normal and malignant hematopoietic cells. Seven of the 19 human HLA class l-restricted mHAgs identified to date exhibit such hematopoietic lineage-restricted presentation. However, a more comprehensive understanding of human mHAgs is urgently needed to define new strategies to enhance GVT while mitigating the significant morbidity and mortality of GVHD. These crucial goals can only be reached by devising innovative, efficient, and economic methods to accelerate the pace of discovery of mHAgs that exhibit selective presentation on hematopoietic cells and relatively high polymorphic frequency. We demonstrate preliminary data indicating that mHAgs can be accurately predicted from high-throughput algorithmic screening of polymorphisms within hematopoietic lineage-restricted genes, and that such candidate mHAgs can be rapidly assessed for their genotype frequency and their ability to be presented and recognized as bona fide mHAgs. We hypothesize that a finite subset of common hematopoietic cell-restricted amino acid polymorphisms functions as HLA-A2 restricted mHAgs involved in eliciting GVT reactivity. Accordingly, the Specific Aims of this proposal are: 1) To select candidate HLA-A2-restricted mHAg epitopes encoded by hematopoietic-specific genes via algorithm-based database searches, and to estimate the allelic frequency of these candidate mHAgs via high-throughput molecular typing. 2) To assess the immunogenicity of the candidate mHAg peptides by the detection of T-cell responses in alloSCT patients using sensitive ex vivo detection methods and in vitro CTL induction experiments with autologous dendritic cells transfected or exogenously pulsed with candidate mHAgs. 3) To assess the natural cell-surface expression of the candidate mHAg peptides, and to validate their hematopoietic-restricted expression at the cellular and RNA levels.